We appreciate assistance from members of the fly community, particularly Dr. Denise Montell, Dr. Lynn Cooley, and Dr. Pernille Rorth, for reagents. We thank Flybase, the Bloomington Drosophila Stock Center, and Developmental Studies Hybridoma Bank for information and providing fly stocks and antibodies, respectively. LM is supported by the Department of Education&#160;Grant, Graduate Assistance in the Areas of National Need (GAANN) training fellowship (P200A120017) and by a NIGMS Initiative for Maximizing Student Development Grant (2 R25-GM55036). A portion of the microscopy work was supported by NSF MRI grant DBI-0722569 and the Keith R. Porter Core Imaging Facility. Research was supported in part by a NSF CAREER Award (1054422) and a Basil O&#8217;Connor Starter Scholar Award from the March of Dimes, both awarded to MSG.

Ovariolesの1。解剖<ol><li>追加された活性乾燥酵母を新鮮なフライ食品のバイアルに約15 2-4日齢の雌のハエや少数の男性を転送します。バイアル用のプラグとして綿を使用し、高湿度を保つために綿に水を数滴を追加します。 </li><li>段8-10卵室の数を最大にするために14〜16時間、25℃のインキュベーター内の場所バイアル。 注：インキュベーション時間は、温度および所望の段階に応じて変化する。 </li><li>次の日のために、必要に応じて解剖メディアを準備し、0.1M KPO 4とNP40ソリューションは、（材料を参照）。 </li><li> CO 2を使用してハエを麻酔し、解剖顕微鏡下に置く。ピペットガラスうつ病スライド内の各キャビティ内に解剖メディアの数滴。 </li><li>一人の女性フライダウン翼を持つテーブルトップと東洋へ約30度の角度でそれぞれの手で鉗子を持ちます。あなたの非利き手では、Aで彼女を把握し、ピンセットを用いて、女性を拾う胸部の近くに腹部のnterior、およびうつ病のスライド（ 図1Aの挿入図）で解剖メディアに彼女を沈める。 </li><li>鉗子の他のペアで、腹部の腹側後部に外骨格を把握し、を貫通。後部端の卵巣をつかみ、その後腹部からそれらを引き出し、うつ病のスライドの反対側に新鮮な培地に入れる。 （ 図1Aの挿入図）。腸を引き離すことは避けてください。死体を捨てる。 </li><li>すべての卵巣を解剖されるまで、他の女性のハエで繰り返します。男性を捨てる。 </li><li>鉗子の1対の、（最大の卵室の近く）の後方端で卵巣を保持し、もう一方の手で最も前方の卵室（germarium）の1を挟ま鉗子のペアを使用。 （ 図1A）。 </li><li>ゆっくりと着実に、卵巣シースの外に卵巣小管チェーンを引っ張る。すべての目的の卵室がRになるまで個別にovariolesを解剖していきemoved。 </li><li>繰り返しますが、すべての解剖卵巣のための1.7から1.8を繰り返します。 </li><li>完了したら、プラスチック製のトランスファーピペットを用いて、0.6ミリリットルチューブにovariolesを移す。 </li><li> ovariolesは、チューブの底に沈殿、定着し、染色のステップに進みましょう。 </li></ol> 2.免疫蛍光（IF）Ovariolesの染色<ol><li>ピペットで注意深くovariolesから解剖メディアを削除する。すべてのovariolesまたは卵室を削除しないように注意してください。 ovariolesと解剖メディアの約50μlのままにしておきます。 </li><li>チューブ中の0.1M KPO 4緩衝液150μlの16％パラホルムアルデヒドの50μlのを追加し、それらを修正するために卵室にソリューションを追加します。 10分間ロッキングしながらインキュベートする。注意：ホルムアルデヒドは有毒である。 </li><li>固定液を除去し、0.5ミリリットルNP40の洗浄緩衝液で2回すすぐ。 </li><li> RTで15分間ずつ2回洗浄する。 </li><li>その後の工程のために、標準的なIFプロトコル15を参照してください。 <ol><li>簡単に言えば、固定後に、追加適切な希釈で一次抗体と4℃でO / Nインキュベートする。 （：200希釈液1）二次抗体を追加し、NP40で数回洗浄する。 </li><li> NP40中で数回洗浄し、その後DAPI（1：1,000）を追加し、10分間、その後の洗浄を繰り返します。プロトコルが完了したIF回、卵室にPBS中50％グリセロールの200μlを添加し、室温で2時間放置。 </li><li> 、溶液を除去し、PBS中70％のグリセロールと交換し、そしてホイルでカバーしています。取り付けの準備が整うまで4ᵒCでサンプルを置きます。一方、ステップ3に進みます。 </li></ol></li></ol>グリセリンゼリースライド3.事前準備<ol><li>スパチュラを使用してゼリーグリセリンの少なくとも4 mlで測定し、ハーフウェイマークにガラス細胞培養管を埋める。 30分間55℃の水浴中でグリセリンゼリーを溶かす。 </li><li> 2顕微鏡スライド上に原液からのグリセロールの小滴、約300μLを、注ぐ。組織を使用して、薄い1aにグリセロールを広めるスライドの各々にわたるヤー。これは、ベースを提供し、ステップ5.6で、グリセリンゼリーを容易に除去することができます。 </li><li>フィルタリング千μlのピペットチップのオフ先端をカット。各顕微鏡スライドにゼリーゆっくり暖かいグリセリンの1,000〜1,500μLを転送するために使用するカットピペットチップ。バブルを防ぐために注意してください。 </li><li>グリセリンゼリースライドを設定するために、室温で5分間座ってみましょう。 </li><li> 60ミリメートル×15ミリメートルのペトリ皿内の各グリセリンゼリースライドを配置し、ラップでカバーしています。 4℃CO / Nにおける場所。必要に応じて、ストアは4℃で最長5日間ゼリースライドグリセリン。 </li></ol> 4.取付エッグチェンバース<ol><li>ステップ2.5、1グリセリンゼリースライド全体で70％のグリセロールで卵室のピペットいくつかの3μlの滴からのサンプルを使用して。すべての卵室は、このスライド上になるまでピペッティング3μlの滴を続行。各ドロップは、少なくとも10卵室が含まれていることを確認してください。一方、55℃の水浴中でグリセリンゼリーの培養管を加熱する。 </li><li>解剖顕微鏡下で卵室とグリセリンゼリースライドを置きます。卵室のみ一滴の視野（ 図1B）になるように倍率を調整する。 </li><li>スプーンのように30ゲージの針を用いて、所望の段階の卵室を拾う。必要な場合には、ナイフなどの針を用いて卵巣小管チェーンから目的の卵室を分離する。 </li><li>イメージングに干渉する可能性デブリを回避するために、前方（ 図1E -1）左に向けて、第グリセリンゼリースライドに卵室を転送する。少なくとも7つの卵室があるまで繰り返し列（ 図1C）に並んだ。 </li><li>すべての目的の卵室は第二グリセリンゼリースライドに転送されるまで、7の新しい列を形成する。 </li><li>組織とねじれの小片を切り取り。軽くそれぞれ1に触れて、卵室から過剰のPBS-グリセロールを吸収するために使用します。卵室を削除しないように注意してください。 </li><li>断つフィルタリング200μlのピペットチップの先端。卵室のすべての列をカバーするために、グリセリンゼリー150μlのを転送するために使用します。 注：列をカバーするのに必要なグリセリンゼリーの量は卵室段と列の数に基づいている。量を調整することができる。 </li><li>グリセリンゼリーのトップ層が完全に（ 図1D）固化するまで取り付けられた卵室は室温で5分間座ってみましょう。 </li><li>置き60ミリメートル×15ミリメートルのペトリ皿に搭載された卵室のスライドとは、ラップでカバーしています。 4℃CO / Nでストア（光退色が懸念される場合には、暗所で行わ）グリセリンゼリー層が一緒に固化することを可能にする。 </li></ol>イメージング用エッグチェンバースの5準備<ol><li>解剖顕微鏡下に取り付けられた卵室のスライドを置きます。卵室の1列のみが視野に入るように倍率を調整する。 </li><li> 45°の角度ミニチュアメスを用いて、straigをカット（卵室の後側に近い）卵室の最初の列の右側に沿ってhtのライン。 （ 図1D-E） </li><li>次に、列の最後の卵室上部の最初の卵室の上下にカット。この時点で、卵室の列には、三方をカットする必要があります。 </li><li>卵室（ 図1E -3）の前方側にできる限り近いマイクロナイフ、列の左側に沿ってスライスし、使用。 </li><li>カットカラムに冷たい1X PBT100μlのピペット。 </li><li>スライド上の残りの部分を残して、卵室のカット列の三辺の周りに余分なグリセリンゼリーを削除し、破棄するように丸い刃へらを使用してください。 </li><li>卵室の前方側で行われたカットでへらを挿入し、主要グリセリンゼリーブロックから列を分離する。 </li><li>反転した（5.8.1）または直立のどちらかのためのサンプル（5.8.2）顕微鏡を準備します。 <ol><li>倒立顕微鏡用：転送トン卵室の前方側を下に向けてカバースリップに卵室の彼が列。グリセリンゼリーのすべての列を削除し、マウントされるまで繰り返します5.2から5.8を繰り返します。乾燥からそれらを防ぐために卵室の各ブロックの上に50％のPBS-グリセロールの滴のカップルを追加します。直接カバースリップ上の画像卵室。 </li><li>正立顕微鏡の場合：前方側を上にして顕微鏡スライドに卵室の列を転送します。グリセリンゼリーのすべての列が削除され、顕微鏡スライドにマウントされるまで繰り返します5.2から5.9を繰り返します。 ＃1 1/2カバーガラスで卵室とカバーブロックの各ブロックの上に50％のPBS-グリセロールの滴のカップルを追加します。 </li></ol></li></ol>

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The authors have no competing financial interests.

ここでは、エンド·オン視点から卵室を開発し、マウントする方法を説明し、画像小。イメージング卵室のための一般的な技術は、横方向の景色のために最適化し、蛍光抗体で染色したときに主に内外方向包細胞の正確な可視化を可能にしている。複数の焦点面を見るのZ-スタックまたは3次元再構成助剤の使用が、それでも楕円形の卵室（ 図2D）の極の細胞内解像度には不十分で...

キイロショウジョウバエの研究は、現象の広い範囲の遺伝的調節に大きな洞察を提供してきました。卵形成は、組織パターニング、細胞極性の変化、細胞周期の切り替え、および翻訳調節1,2,3,4を含む多くの異なる発生プロセスを調査する扱いやすい方法を提供するので、特に、卵の開発に関する広範な研究があった。卵形成時の重要な形態形成のイベントは（5で検討）ボーダー細胞と呼ばれる細胞のセットの仕様、運動性の獲得、および移行である。細胞移動するので、動物の形態形成の重要な特徴であり、このプロセスの遺伝的調節は、よく保存されているため、ハエで決定メカニズムは、他の文脈において重要である可能性が高い。従って、我々は国境細胞遊走の分子制御を研究している。私たちの研究のために、我々は、発展途上の卵の前極を観察する新しい方法を開発した、ここで、境界セルは、それらが詳細に開発かを検討するために、発生する。 卵巣内では、様々な発生段階の卵室が薄いシース（ 図1A）に収められていovariolesと呼ばれるチェーンに沿って存在する。各卵室は、1個の卵を形成するために移動します。卵形成の間に、いくつかの異なる細胞型が協調して開発しなければならない。 D.ショウジョウバエの卵室は、単層濾胞上皮6に囲まれた1卵母細胞を含む16生殖細胞から成る。特殊化した細胞の小さな数は、上皮の前部および後部の極で発生する、極性細胞と呼ばれる。 6-8ボーダー細胞は極性細胞5,7により誘導された卵室の前方上皮、に由来する。中期卵形成（ステージ9）では、ボーダー細胞は、その隣人から切り離し、卵室5,7の後部に卵母細胞に到達するために看護師セル間で移動する。この移動は、達成されなければならない境界セルは、この集団の細胞遊走の種類て、2つの非運動性極性細胞の周囲のクラスタに残っている。境界セルクラスタの移行を成功さは受精のために必要がある、卵の殻の卵門の適切な発展を保証します。 前極細胞は、シグナル伝達カスケードを活性化することにより、境界細胞の運命を指示する。ポーラー細胞は卵母細胞の発達8,9の段階8の間に包細胞を、隣接上、Domeless（DOME）、膜貫通受容体に結合するサイトカイン、不対（UPD）を、分泌する。 UPDの結合は、転写のシグナルトランスデューサーおよび活性化因子（STAT）8,10,11をリン酸化するヤヌスチロシンキナーゼ（JAK）を引き起こす。 STATはその後転写を活性化するために、核に移動します。遅い境界細胞（SLBO）STATの直接的な転写ターゲットであり、また、境界細胞遊走12に必要とされる転写因子である。卵室の側面図は、Tを示している帽子STAT活性は、前方上皮8,11,13-全体の勾配で規制されている。極性細胞に最も近い包細胞は、このように、彼らは国境細胞になると、隣接する生殖系列の組織に侵入、活性化したSTATの最高レベルを持っている。 ボーダー細胞が内で指定されているかを理解し、上皮から切り離すために、我々は組織が編成されているかを観察する必要があります。我々は、前·オン視点から卵室を表示する場合は、極性細胞周囲の毛包細胞中のSTAT活動の放射状の対称性を期待する。エンド上のビューは、より正確に前と異なる焦点面で細胞を比較するより剥離時の膜タンパク質と細胞間のインタフェースの違いを示すであろう。卵室が楕円形と茎細胞によって互いに取り付けられているので、それらが困難前方アーキテクチャを観察すること、横方向にスライド上に沈降する。このように、卵室の極にある細胞に関する多くの情報があります側面図から推測されて。一部の情報は、光退色光セクション、光散乱、のアルゴリズムの3-D再構成、及びZ軸方向の分解能の悪い限界を介して取得することができるが、超解像顕微鏡14のような高価な技術が存在しない場合、この情報は、以下の詳細および信頼性を高める。セクションベースのイメージング（例えば、電子顕微鏡またはミクロトーム切片）他の種類のアーチファクトのために可能性を増加させる、脱水を含む組織の大規模な操作を必要とする。従って、我々は画像Dに新しい方法を開発キイロ卵室直立している。この方法は、すでに（ 審査中で、代表的な結果とマニングらを参照）運動性の細胞が運命づけられているかの解明に有用であることが証明され、卵形成の他の側面の研究でより広く貴重である可能性が高いしている。

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			<td height="40" style="height:40px;width:348px;">Name of Reagent/ Equipment</td>
			<td style="width:279px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:283px;">Comments/Description</td>
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		<tr height="96">
			<td height="96" style="height:96px;width:348px;">0.1 M Potassium phosphate Buffer (KPO4 Buffer)</td>
			<td style="width:279px;"></td>
			<td style="width:119px;"></td>
			<td style="width:283px;">Add 3.1 g of NaH2PO4&#8226;H2O and 10.9 g of Na2HPO4 (anhydrous) to distilled H2O to make a volume of 1 L. The pH of the final solution will be 7.4. This buffer can be stored for up to 1 mo at 4&#176;C.</td>
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			<td height="25" style="height:25px;width:348px;">16% Paraformaldehyde aqueous solution, EM grade</td>
			<td style="width:279px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">15710</td>
			<td>methanol-free to preserve GFP fluorescence</td>
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			<td height="20" style="height:20px;width:348px;">30G x 1/2 inch needle&#160;</td>
			<td style="width:279px;">VWR</td>
			<td style="width:119px;">BD305106</td>
			<td>Regular bevel</td>
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			<td height="22" style="height:22px;width:348px;">Active Dry Yeast</td>
			<td style="width:279px;">Genesee Scientific</td>
			<td>62-103</td>
			<td>To fatten female flies</td>
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			<td height="20" style="height:20px;width:348px;">Bovine Serum Albumin</td>
			<td style="width:279px;">PAA-cell culture company</td>
			<td style="width:119px;">A15-701</td>
			<td>Used in NP40 Wash Buffer</td>
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		<tr height="39">
			<td height="39" style="height:39px;width:348px;">Dumont #5 Forceps</td>
			<td style="width:279px;">Fine Science Tools</td>
			<td style="width:119px;">11295-10</td>
			<td style="width:283px;">Dumostar alloy, biologie tip; sharp tips are essential for ovariole dissection</td>
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		<tr height="39">
			<td height="39" style="height:39px;width:348px;">DAPI (4&#8242;,6-Diamidino-2-phenylindole dihydrochloride)</td>
			<td style="width:279px;">Sigma Aldrich</td>
			<td style="width:119px;">D9542</td>
			<td style="width:283px;">5mg/ml stock solution&#160;&#160;</td>
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			<td height="39" style="height:39px;width:348px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:279px;">Life Technologies</td>
			<td style="width:119px;">16140-071</td>
			<td style="width:283px;">Added to supplement dissection medium (10%)&#160;</td>
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			<td>47729-576</td>
			<td>14mL</td>
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			<td height="20" style="height:20px;width:348px;">Glass Depression Slides</td>
			<td style="width:279px;">VWR</td>
			<td>470019-020</td>
			<td>1.2 mm Thick, Double Cavity</td>
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			<td>17998-10</td>
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			<td style="width:119px;">IB15760</td>
			<td>70% in PBS</td>
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			<td height="39" style="height:39px;width:348px;">IGEPAL CA-630</td>
			<td style="width:279px;">Sigma Aldrich</td>
			<td style="width:119px;">I3021-500ML</td>
			<td style="width:283px;">(interchangable for Nonidet P-40)&#160; Used in NP40 Wash Buffer&#160;</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Leica Fluorescent Stereoscope&#160;</td>
			<td style="width:279px;">Leica Microsystems</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Leica SP5 Confocal Microscope</td>
			<td style="width:279px;">Leica Microsystems</td>
			<td style="width:119px;"></td>
			<td>40x/0.55NA dry objective&#160;</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Micro spatula&#160;</td>
			<td style="width:279px;">VWR</td>
			<td style="width:119px;">82027-518</td>
			<td>Stainless steel</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Microknife&#160;</td>
			<td style="width:279px;">Roboz Surgical Instrument Company</td>
			<td style="width:119px;">37-7546</td>
			<td>45&#7506; angle</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Microscope Slide</td>
			<td style="width:279px;">VWR</td>
			<td style="width:119px;">16004-368</td>
			<td>75x25x1 mm</td>
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		<tr height="58">
			<td height="58" style="height:58px;width:348px;">NP40 Wash Buffer</td>
			<td style="width:279px;"></td>
			<td style="width:119px;"></td>
			<td style="width:283px;">50mM TRIS-HCL, 150mM NaCl, 0.5% Ipegal, 1mg/ml BSA, and 0.02% sodium azide</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Penicillin-Streptomycin-Glutamine</td>
			<td style="width:279px;">Life Technologies</td>
			<td>10378-016</td>
			<td>Added to supplement dissection medium (0.6X)</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Petridish- Polysterine, sterile</td>
			<td style="width:279px;">VWR</td>
			<td>82050-548</td>
			<td>60Wx15H mm</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Phosphate Buffer Saline</td>
			<td style="width:279px;">Sigma Aldrich</td>
			<td style="width:119px;">P3813-10PAK</td>
			<td>10 packs of Powder</td>
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			<td height="20" style="height:20px;width:348px;">Potassium phosphate dibasic</td>
			<td style="width:279px;">Sigma Aldrich</td>
			<td style="width:119px;">P3786-500G</td>
			<td>Used in 0.1M KPO4 Buffer&#160;</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Potassium phosphate monobasic</td>
			<td style="width:279px;">Sigma Aldrich</td>
			<td style="width:119px;">P9791-500G</td>
			<td>Used in 0.1M KPO4 Buffer&#160;</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Schneider&#8217;s Insect Medium</td>
			<td style="width:279px;">Life Technologies</td>
			<td style="width:119px;"> 
			11720018</td>
			<td>With L-glutamine and sodium bicarbonate</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:348px;">Sodium azide</td>
			<td style="width:279px;">Sigma Aldrich</td>
			<td style="width:119px;">S2002-25G</td>
			<td>Used in fluorescent antibody staining</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Sodium chloride</td>
			<td style="width:279px;">Sigma Aldrich</td>
			<td style="width:119px;">S3014-500G</td>
			<td>Used in NP40 Wash Buffer</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:348px;">TRIS-HCL</td>
			<td style="width:279px;">IBI Scientific</td>
			<td style="width:119px;">IB70144</td>
			<td>1M TRIS-HCL, pH 7.4</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Volocity 3D Image Analysis Software</td>
			<td style="width:279px;">PerkinElmer</td>
			<td style="width:119px;"></td>
			<td>For processing confocal Z-stacks</td>
		</tr>
	</tbody>
</table>

upright imaging of drosophila egg chambers

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in architecture, is well-characterized, and is amenable to genetic analysis. Each egg chamber consists of germ-line cells surrounded by a single epithelial layer of somatic follicle cells. Subsets of follicle cells undergo differentiation during specific stages to become several different cell types. Standard techniques primarily allow for a lateral view of egg chambers, and therefore a limited view of follicle cell organization and identity. The upright imaging protocol describes a mounting technique that enables a novel, vertical view of egg chambers with a standard confocal microscope. Samples are first mounted between two layers of glycerin jelly in a lateral (horizontal) position on a glass microscope slide. The jelly with encased egg chambers is then cut into blocks, transferred to a coverslip, and flipped to position egg chambers upright. Mounted egg chambers can be imaged on either an upright or an inverted confocal microscope. This technique enables the study of follicle cell specification, organization, molecular markers, and egg development with new detail and from a new perspective.

直立イメージング法は、私たちはステージ8の前方濾胞上皮に直接卵胞細胞の運命のための一般的なマーカーを細胞の組織を見ることができ、目の不在（EYA）タンパク質だけでなく、核DNAマーカーDAPIは、さえ発現を示した細胞のこの分野全体で、すべてのセルが同様の染色強度（ 図2B」）で見ることができることを実証した。サイトカインUPDに応答して調節されるタンパク質は、...

Watch this Scientific Journal Video about Upright Imaging of Drosophila Egg Chambers at JoVE.com

Upright Imaging of Drosophila Egg Chambers

The upright imaging method described in this protocol allows for the detailed visualization of the poles of a developing Drosophila melanogaster egg. This end-on view provides a new perspective into the arrangements and morphologies of multiple cell types in the follicular epithelium.

のアップライトイメージング<em&gt;ショウジョウバエ</em&gt;エッグチェンバース

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in ...

upright-imaging-of-drosophila-egg-chambers

Research

JoVE Journal

Developmental Biology

Upright Imaging of Drosophila Egg Chambers

7.5K ビュー.  University of Maryland Baltimore County. The upright imaging method described in this protocol allows for the detailed visualization of the poles of a developing Drosophila melanogaster egg. This end-on view provides a new perspective into the arrangements and morphologies of multiple cell types in the follicular epithelium.

ビデオ: Upright Imaging of Drosophila Egg Chambers

Live-imaging of the Drosophila Pupal Eye

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track during live cell imaging. These processes include: retinal rotation, cell growth and organismal movement. Additionally, irregularities in the topology of the epithelium, including subtle bumps and folds often introduced as the pupa is prepared for imaging, make it challenging to acquire in-focus images of more than a few ommatidia in a single focal plane. The workflow outlined here remedies these issues, allowing easy analysis of cellular processes during Drosophila pupal eye development. Appropriately-staged pupae are arranged in an imaging rig that can be easily assembled in most laboratories. Ubiquitin-DE-Cadherin:GFP and GMR-GAL4&#8211;driven UAS-&#945;-catenin:GFP are used to visualize cell boundaries in the eye epithelium 1-3. After deconvolution is applied to fluorescent images captured at multiple focal planes, maximum projection images are generated for each time point and enhanced using image editing software. Alignment algorithms are used to quickly stabilize superfluous motion, making individual cell behavior easier to track.

Live-imaging of the Drosophila Pupal Eye

This protocol presents an efficient method for imaging the live Drosophila pupal eye neuroepithelium. This method compensates for tissue movement and uneven topology, enhances visualization of cell boundaries through the use of multiple GFP-tagged junction proteins, and uses an easily-assembled imaging rig.

のライブイメージング<em&gt;ショウジョウバエ</em&gt;蛹アイ

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track ...

発生生物学

Imaging Calcium in Drosophila at Egg Activation

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. One aspect of egg activation, conserved across all organisms examined, is a change in the intracellular concentration of calcium (Ca2+) often termed a 'Ca2+ wave'. While the speed and number of oscillations of the Ca2+ wave varies between species, the change in intracellular Ca2+ is key in bringing about essential events for embryonic development. These changes include resumption of the cell cycle, mRNA regulation, cortical granule exocytosis, and rearrangement of the cytoskeleton.
In the mature Drosophila egg, activation occurs in the female oviduct prior to fertilization, initiating a series of Ca2+-dependent events. Here we present a protocol for imaging the Ca2+ wave in Drosophila. This approach provides a manipulable model system to interrogate the mechanism of the Ca2+ wave and the downstream changes associated with it.

Imaging Calcium in Drosophila at Egg Activation

This article describes an adaptable ex vivo protocol for visualizing Ca2+ during egg activation in Drosophila.

内イメージングカルシウム<em&gt;ショウジョウバエ</em&gt;卵活性化で

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. ...

Long-term Live Imaging of Drosophila Eye Disc

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have been used to study many biological processes, such as cell proliferation, differentiation, growth, migration, apoptosis, competition, cell-cell signaling, and compartmental boundary formation. However, methods for the long-term ex vivo culture and live imaging of the imaginal discs have not been satisfactory, despite many efforts. Recently, we developed a method for the long-term ex vivo culture and live imaging of imaginal discs for up to 18 h. In addition to using a high insulin concentration in the culture medium, a low-melting agarose was also used to embed the disc to prevent it from drifting during the imaging period. This report uses the eye-antennal discs as an example. Photoreceptor R3/4-specific m&#948;0.5-Ga4 expression was followed to demonstrate that photoreceptor differentiation and ommatidial rotation can be observed during a 10 h live imaging period. This is a detailed protocol describing this simple method.

Long-term Live Imaging of Drosophila Eye Disc

This protocol describes a detailed method for the long-term ex vivo culture and live imaging of a Drosophila imaginal disc. It demonstrates photoreceptor differentiation and ommatidial rotation within the 10 h period of live imaging of the eye disc. The protocol is simple and does not require expensive setup.

長期のライブイメージング<em&gt;ショウジョウバエ</em&gt;アイディスク

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have ...

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from animals to humans. Therefore, the powerful Drosophila model system can be used to study concepts of muscle-tendon development that can also be applied to human muscle biology. Here, we describe in detail how morphogenesis of the adult muscle-tendon system can be easily imaged in living, developing Drosophila pupae. Hence, the method allows investigating proteins, cells and tissues in their physiological environment. In addition to a step-by-step protocol with helpful tips, we provide a comprehensive overview of fluorescently tagged marker proteins that are suitable for studying the muscle-tendon system. To highlight the versatile applications of the protocol, we show example movies ranging from visualization of long-term morphogenetic events &#8211; occurring on the time scale of hours and days &#8211; to visualization of short-term dynamic processes like muscle twitching occurring on time scale of seconds. Taken together, this protocol should enable the reader to design and perform live-imaging experiments for investigating muscle-tendon morphogenesis in the intact organism.

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Here, we present an easy-to-use and versatile method to perform live imaging of developmental processes in general and muscle-tendon morphogenesis in particular in living Drosophila pupae.

生体内でショウジョウバエの蛹の筋腱の形態形成の観察

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from ...

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ellipsoid shape during development. This developmental process is called oogenesis and is crucial to generating functional mature eggs to secure the next fly generation. For these reasons, egg chambers have served as an ideal and relevant model to understand animal organ development.
Several in vitro culturing protocols have been developed, but there are several disadvantages to these protocols. One involves the application of various covers that exert an artificial pressure on the imaged egg chambers in order to immobilize them and to increase the imaged acquisition plane of the circumferential surface of the analyzed egg chambers. Such an approach may negatively influence the behavior of the thin actomyosin machinery that generates the power to rotate egg chambers around their longer axis.
Thus, to overcome this limitation, we culture Drosophila egg chambers freely in the media in order to reliably analyze actomyosin machinery along the circumference of egg chambers. In the first part of the protocol, we provide a manual detailing how to analyze the actomyosin machinery in a limited acquisition plane at the local cellular scale (up to 15 cells). In the second part of the protocol, we provide users with a new Fiji-based plugin that allows the simple extraction of a defined thin layer of the egg chambers&#8217; circumferential surface. The following protocol then describes how to analyze actomyosin signals at the tissue scale (&#62;50 cells). Finally, we pinpoint the limitations of these approaches at both the local cellular and tissue scales and discuss its potential future development and possible applications.

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

This protocol provides a Fiji-based, user-friendly methodology along with straightforward instructions explaining how to reliably analyze actomyosin behavior in individual cells and curved epithelial tissues. No programming skills are required to follow the tutorial; all steps are performed in a semi-interactive manner using the graphical user interface of Fiji and associated plugins.

培養ショウジョウバエ卵室のタイムラプスムービーを用いた局所細胞および組織スケールにおけるアトミオシンダイナミクスの解析

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ...

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits are required to control this behavior. Failure to produce the rhythmic pattern of contractions can be indicative of neurological abnormalities. We previously found that defects in protein O-mannosylation, a posttranslational protein modification, affect the axon morphology of sensory neurons and result in abnormal coordinated muscle contractions in embryos. Here, we present a relatively simple method for recording and analyzing the pattern of peristaltic muscle contractions by live imaging of late stage embryos up to the point of hatching, which we used to characterize the muscle contraction phenotype of protein O-mannosyltransferase mutants. Data obtained from these recordings can be used to analyze muscle contraction waves, including frequency, direction of propagation and relative amplitude of muscle contractions at different body segments. We have also examined body posture and taken advantage of a fluorescent marker expressed specifically in muscles to accurately determine the position of the embryo midline. A similar approach can also be utilized to study various other behaviors during development, such as embryo rolling and hatching.

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Here, we present a method to record embryonic muscle contractions in Drosophila embryos in a non-invasive and detail-oriented manner.

ショウジョウバエ胚における筋収縮のライブイメージングと解析

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits ...

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the number of novel cell lines is expected to increase. The DGRC aims to familiarize researchers with using Drosophila cell lines as an experimental tool to complement and drive their research agenda. Procedures for working with a variety of Drosophila cell lines with distinct characteristics are provided, including protocols for thawing, culturing, and cryopreserving cell lines. Importantly, this publication demonstrates the best practices required to work with Drosophila cell lines to minimize the risk of contaminations from adventitious microorganisms or from other cell lines. Researchers who become familiar with these procedures will be able to delve into the many applications that use Drosophila cultured cells including biochemistry, cell biology and functional genomics.

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Drosophila cell lines are important reagents for both fundamental and biomedical research. This article provides protocols for thawing, subculturing, and the cryopreservation of commonly used Drosophila cell lines to assist researchers in incorporating the use of these reagents in their research.

解凍、養殖、ショウジョウバエ細胞をトウキョウ

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the ...

Human Egg Maturity Assessment and Its Clinical Application

The optimal timing of intracytoplasmic sperm injection (ICSI) is of a serious concern for fertility programs because untimely sperm entry diminishes the egg&#39;s developmental competence. Presence of the first polar body (PB) together with the meiotic spindle indicates completion of the oocyte maturation and the egg&#39;s readiness for fertilization. In clinical practice, it is customary to assume that all oocytes displaying a PB are mature metaphase (MII) oocytes. However, PB extrusion precedes the formation of the bipolar MII spindle. This asynchrony makes the mere presence of PB an unreliable marker of oocyte maturity. Noninvasive spindle imaging using polarized light microscopy (PLM) allows quick and easy inspection of whether the PB-displaying oocyte actually reassembled a meiotic spindle prior to ICSI. Here, we present a standard protocol to perform human egg maturity assessment in the clinical laboratory. We also show how to optimize the time of ICSI with respect to the oocyte&#39;s developmental stage in order to prevent premature sperm injection of late-maturing oocytes. Using this approach, even immature oocytes extruding PB in vitro can be clinically utilized. Affirmation that MII spindle is present prior to sperm injection and individual adjustment of the time of ICSI is particularly important in poor prognosis in-vitro fertilization (IVF) cycles with a low number of oocytes available for fertilization.

We provide an outline of the clinical protocol for non-invasive assessment of human egg maturity using polarized light microscopy.

ヒト卵成熟度評価とその臨床応用

The optimal timing of intracytoplasmic sperm injection (ICSI) is of a serious concern for fertility programs because untimely sperm entry diminishes the ...

Imaging Dpp Release from a Drosophila Wing Disc

The transforming Growth Factor-beta (TGF-&#946;) superfamily is essential for early embryonic patterning and development of adult structures in multicellular organisms. The TGF-&#946; superfamily includes TGF-&#946;, bone morphogenetic protein (BMPs), Activins, Growth and Differentiation Factors, and Nodals. It has long been known that the amount of ligand exposed to cells is important for its effects. It was thought that long-range concentration gradients set up embryonic pattern. However, recently it has become clear that the timing of exposure to these ligands is also important for their downstream transcriptional consequences. A TGF-&#946; superfamily ligand cannot have a developmental consequence until it is released from the cell in which it was produced. Until recently, it was difficult to determine when these ligands were released from cells. Here we show how to measure the release of a Drosophila BMP called Decapentaplegic (Dpp) from the cells of the wing primordium or wing disc. This method could be modified for other systems or signaling ligands.

Imaging Dpp Release from a Drosophila Wing Disc

The timing of exposure to ligands may impact their developmental consequences. Here we show how to image release of a Drosophila bone morphogenetic protein (BMP) called Dpp from cells of the wing disc.

ショウジョウバレウィングディスクからのイメージングDppリリース

The transforming Growth Factor-beta (TGF-&#946;) superfamily is essential for early embryonic patterning and development of adult structures in ...

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

Biomedical imaging tools permit investigation of molecular mechanisms across spatial scales, from genes to organisms. Drosophila melanogaster, a well-characterized model organism, has benefited from the use of light and electron microscopy to understand gene function at the level of cells and tissues. The application of imaging platforms that allow for an understanding of gene function at the level of the entire intact organism would further enhance our knowledge of genetic mechanisms. Here a whole animal imaging method is presented that outlines the steps needed to visualize Drosophila at any developmental stage using microcomputed tomography (&#181;-CT). The advantages of &#181;-CT include commercially available instrumentation and minimal hands-on time to produce accurate 3D information at micron-level resolution without the need for tissue dissection or clearing methods. Paired with software that accelerate image analysis and 3D rendering, detailed morphometric analysis of any tissue or organ system can be performed to better understand mechanisms of development, physiology, and anatomy for both descriptive and hypothesis testing studies. By utilizing an imaging workflow that incorporates the use of electron microscopy, light microscopy, and &#181;-CT, a thorough evaluation of gene function can be performed, thus furthering the usefulness of this powerful model organism.

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

A protocol is presented that allows for the visualization of intact Drosophila melanogaster at any stage of development using microcomputed tomography.